Abstract #887: Genetic and expression changes associated with ellipticine-resistance in high-risk neuroblastoma cell line

Abstract

Neuroblastoma is the most frequent extra-cranial tumor in children and is a major cause of death from neoplasia in infancy. One of the main causes of chemotherapy failure is the rise of drug-resistance. The aim of our work was a genetic and expression study of neuroblastoma cell line resistant to ellipticine (UKF-NB-4^ELLI) compared to sensitive cell line (UKF-NB-4). Ellipticine is cytostatic drug used in metastatic breast cancer and AML. UKF-NB-4^ELLI was prepared by long-term cultivation of UKF-NB-4 cell line with increasing concentrations of ellipticine. UKF-NB-4^ELLI has decreased tumorigenicity and slowed growth in vivo in nunu mice in comparison to UKF-NB-4. The prevalent mechanisms of ellipticine antitumor activities were suggested to be its intercalation into DNA and inhibition of topoisomerase II. Ellipticine also covalently binds to DNA after being enzymatically activated by cytochromes P450 (CYP) or peroxidases, suggesting a third possible mechanism of action. We used comparative genomic hybridization to compare DNA from UKF-NB-4 with that of normal human DNA and DNA from UKF-NB-4^ELLI with that of the parent UKF-NB-4. Fluorescence in situ hybridization was used for verification of genetic changes. For expression profile we used expression-microarray and for verification expression changes RT PCR. In UKF-NB-4, we identified trisomy in the 7^th chromosome with a gain of 7q21, where genes of MDR1 (protein product P-glycoprotein - P-gp) and CYP3A are located. The UKF-NB-4^ELLI has trisomy of the 7^th chromosome with no other changes (loss of 7q21 gain). UKF-NB-4^DOXO, resistant to doxorubicin, has a gain of MDR1 gene and a high P-gp expression. In the UKF-NB-4^ELLI, no differences in expression of P-gp compared to UKF-NB-4 were found. Also genes of the two ellipticine-oxidizing cytochromes P450, CYP1A1 and CYP1B1, localized in area 15q22-24 and 2p21 respectively, did not change. Together it suggests that resistance to ellipticine in UKF-NB-4^ELLI is not caused by a change in metabolic or efflux activity. However, UK-NB-4 has a gain of topoisomerase II (TOPO2), while UKF-NB-4^ELLI has lost one gene copy and a variety of genes corresponding to trisomy of chromosome 17. Expression of TOPO2 was also decreased. Another ellipticine-activating enzyme is myeloperoxidase (located in 17q23). UKF-NB-4 has a gain of 17q23. UKF-NB-4^ELLI has lost one copy, but still has a 17q23 gain. We found three changes that might explain the drug-resistance to ellipticine. The first one is deletion of BAX gene (19q13.33), that codes apoptotic protein Bax, the second one is increased expression of antiapoptotic protein Bcl-2 and the third one is decreased expression of TOPO2. Grant support: GAUK 7926/2007, IGA MZ 1A8696-4/2005 and MSM0021620813.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 887.